From cells to organs with lasers

Transcription

1 From cells to organs with lasers Halina Rubinsztein-Dunlop M. J. Friese,, G. Knöner ner,, T. Nieminen,, S. Parkin, J. Ross, W. Singer, C. Talbot, N.R. Heckenberg Centre for Biophotonics and Laser Science, School of Physical Sciences University of Queensland, Brisbane, Australia Biophotonics in Australia, February 2006 Biophotonics Biophotonics is the science of generating and harnessing light (photons) to image, detect and manipulate biological materials At the interface of physical, biological and medical sciences The Institute for Lasers, Photonics and Biophotonics, University at Buffalo NSF Centre for Biophotonics, Science and Technology, UC Davis Bio-X, Stanford Division of Biophysics and Imaging, Ontario Cancer Institute Beckman Laser Institute, Irvine 2 1

2 OUR EXPERTISE Fundamental Laser Science Laser Manipulation Eg. Laser Tweezers Light activated processes Functional Biopolymer Spectroscopy Eg. Melanin spectroscopy Biomedical & Clinical Applications Eg. Lasers in Dentistry Biomedical Imaging Plus expertise in tissue engineering.,through our Affiliates 3 Examples of Biophotonics projects Hyperpolarised Gases for MRI Imaging Light activated processes Laser micromanipulation and examples of applications 4 2

3 Hyperpolarized Gas MR Imaging Dynamic images of the human lung during inhalation and expiration of 3 He The use of hyperpolarized 3 He and 129 Xe for imaging air spaces and certain tissues in humans. Traditional MRI techniques derive images from hydrogen. In places such as the lungs where hydrogen is not so abundant, imaging is difficult using these techniques. 5 Hyperpolarized Gas MR Imaging 3 He and 129 Xe polarisation processes are both based on the spin exchange optical pumping technique Time necessary to hyperpolarize the noble gas as well as the amount of gas produced and the process used to collect it is different. 6 3

4 Spin Exchange Optical Pumping (SEOP) SEOP method can be used for both noble gases Same Rb absorption line same laser λ = nm Otherwise, process is quite different for 3 He and 129 Xe 7 Variables for SEOP Gas Pressure - Affects the Rb absorption line - Changes laser requirements Temperature - Rb number density Gas mixture - Determined by collisional cross-section Time in the Spin Exchange region T1 of the vessel Magnetic field, photon quality 8 4

5 HP Gas Polarizer Pyrex cell containing 98% 3 He, 2% N 2, Rb Helmholtz Coils create uniform magnetic field laser Laser polarization of Rb Laser frequency narrowing apparatus Oven 5P ½ collisional mixing m j =-½ σ + m j =½ Collisional transfer to He B r 5S ½ 1/3 2/3 Radiative decays 9 Laser Frequency Narrowing why bother? Maximize 3 He Polarization Rate Maximize spin exchange collisions with Rb Density of species Maximize Rb polarization Quality of the laser light Polarization Spectrum Laser power Absolute Power Spectral power 10 5

6 Improving Spectral Power Density Free running laser spectrum spans 3-5 nm Rb absorption ~0.5 nm Power (W) Putting all the laser power in a narrow band would increase spectral power density and thus Rb polarization rate λ (nm) Centre λ (Rb resonance, nm) 11 3 He : progress Produced HP 3 He Achieved ~15% polarization on first attempt 12 6

7 Images The cell 15 % polarisation 13 Imaging balloon rat s lung 14 7

8 Light modified fluorophore transporters The interaction of light with some organisms can drive the transport of solutes across the cellular membrane. The transport of these solutes may be measured by patch clamping methods by optical microscopy epifluorescent bright field microscopy imaging of calcium ions using two photon excitation at an electronic resonance and monitoring the fluorescence emission of the ion. 15 A hybrid of single-photon and multi-photon imaging Decoupling of the membrane transporter and the excitation of fluorescence from the fluorophore. The system allows simultaneous and independent activation of the light induced membrane transport and imaging of the fluorophore. Giardia 16 8

9 Uptake of the fluorophore Justin Ross Confocal Microscopy, light-activated pump action of cells 17 More from the Confocal 5 µm Justin Ross The localisation of Rhodamine 123 in Geardia trophozoites 18 9

10 A hybrid of single-photon and multi-photon imaging fs pulse Excitation Light Fluorescence Em ission Light Ac tivation Light Em issio n Fite rs PMT Confocal Scanner Unit Dichroic Mirror Ti:Sapph Steering Mirrors Nd:YVG Objec tive lens Stage Sa mple Condenser lens Hg Lamp Activation Filte r 19 Spectral Response of light induced uptake of Rh123 by Geardia Spectral Response of G. duodenalis Uptake of Rhodamine Strain WB1B Gaussian Fit of Data Rhodamine 123 Emission Rhodamine 123 Excitation 70 Change in Fluorescence (µm/µj) Wavelength (nm) 20 10

11 Influx and Efflux Justin Ross 21 Influx and Efflux Influx and Efflux of Dye from Giardia 35 Dye Concentration in Cell Rate of Dye Influx Dye Concentration (um) Concentration Change (um/s) Time (s)

12 Optical micromanipulation - using light to make: Laser tweezers Laser scissors Laser catapult Laser screwdriver Optical spanner (wrench) (Simpson et al, Opt Lett,1997) optical torque wrench measures torque as it is applied 23 Typical Laser Tweezers setup Can trap and manipulate high index particles in water Typical: 1µm radius, 1pN/mW 24 12

13 Optical Trapping Ray optics model Force on a point dipole Force results from exchange of momentum with beam 25 Optical Force F = np c Q opt n - refractive of the trapped object, P - incident laser power Q opt - efficiency factor Fdrag = 6πη av max trap against viscous drag or movement Q v a c trans = 6πη max np 26 13

14 2 mm polystyrene spheres Gregor Knöner 27 Trapping and manipulation of 2 µm polystyrene spheres Gregor Knöner 28 14

15 Trapping of a macrophage Gregor Knöner 29 Studies of the dynamic structure of individual nucleosomes (DNA organised into nucleosomes) Stretching nucleosomal arrays with feedback-enhanced optical trap. Brent D. Brower-Toland et al. PNAS,

16 Rotating Tweezers Creating torque and measuring it as it is applied Asymmetric particles in laser beam Special laser beams Multiple traps 31 Optical Angular Momentum I Helical wavefront ( orbital a.m.) optical vortex singular beam Gauss-Laguerre GL pl Gaussian beam AM = integer x ћ per photon l = 1 Phase singularity on axis, dark spot 32 generated by laser, or phase plate or hologram 16

17 Optical Angular Momentum orbital Helical wavefront p r l = 1 l = 3 p φ L= r x p Associated with spatial distribution 33 Laser beam with orbital a.m. Helical wavefronts l = 3 Absorbing particle He et al., PRL (1995) Not useful because of heating 34 17

18 Optical Angular Momentum II Spin Circularly polarised light.ћ per photon (spin) 35 Transfer of AM to a waveplate CaCO 3 particles in H 2 O are 3-D trapped in polarised light They either rotate continuously or align to a particular orientation Plane polarised laser beam λ/4 or λ/2 plate high N.A. lens In linear light, their orientation is controllable In elliptical light, their rotation frequency is controllable. Optical torque Trapped calcite crystal Drag torque Friese et al., Nature

19 Transfer of angular momentum of light alignment Optical axis of crystal aligns to electric field vector 37 Calcite crystal rotates in circularly polarised light Friese et al, Nature 394, ,

20 All optically driven micromachine elements 39 Friese et al, Appl. Phys. Lett. 78, 1, 2001 Micro-rheology Circularly polarised light h per photon λ/4 waveplate zero Try to maintain circular symmetry to avoid orbital 40 A.M. 20

21 Making better probes Spherical CaCO 3 Crystals optical image Better probe particles - smooth rotation -3D trapping SEM image Alexis Bishop et al, PRL Theory α => µ = Where α = shear stress Viscosity ratio of γ stress to strain γ = shear rate σp Where Applied Torque => τ R = σ = change in polarisation or α ω P = laser power We measure viscosity µ = by applying Where α a = stress shear stress ω = optical to frequency the 3 Where Drag fluid using Torque a => sphere τ D = γrotated γ 8πµ a Ωby circularly µ = viscosity shear of rate liquid polarised The response light of with the a fluid known torque a = sphere s radius Ω = frequency of rotation Applied Viscous Torque drag = torque Drag Torque Where σ = change in viscosity of the surrounding 3 τ σp liquid polarisation D = 8τ πµ R = a Ω Surrounding Liquid P = laser power creating ω σp quite a ω = optical frequency stir! Where µ = viscosity of surrounding µ = liquid a = sphere s radius 3 8πa Ω = frequency of rotation Ωω This sphere s 42 21

22 CaCO 3 sphere spinning inside an artificial liposome (15µm dia) 10µm Alexis Bishop et al, PRL Four vaterite spheres rotating inside an artificial liposome 10µm 44 22

23 Laser tweezers, scissors and wrench Gregor Knöner 45 Fluid-Coupled MechanoTransduction Flow-Coupled Shear Stress Elliot Botvinick, Gregor Knöner et al 46 23

24 Optically driven micromachines Simon Parkin 47 Optically driven micromachines Gregor Knöner 48 24

25 Two-photon photopolimerization Two-photon initiated photopolymerization for threedimensional fabrication SEM image of a micro-bull sculpture. Kawata, Japan Exposure Apparatus lamp millennia tsunami attenuator objective shutter condenser 3d piezo stage imaging optics CCD M1 λ 2 λ 4 Nd:YAG fiber laser subsequent production and trapping Ti:Sapphire laser, 1 W, 80fs, 780nm for production of structure 50 25

26 Two-photon photopolimerization at UQ 15 mm 21 mm Julien Higuet 51 Microhologram for Lab-on-Chip on axis phase hologram transfer of angular momentum to linear polarized Gaussian beam 52 26

27 SEM of phase hologram 8 mm 53 Angular momentum transfer polystyrene spheres, d = 2.1 µm incident beam: Gaussian, linear polarized linear polarized beam no angular momentum λ/4 waveplate circularly polarized spin angular momentum transfer to anisotropic particle TEM 00, linear pol. no angular momentum phase hologram LG 04, helical wavefront orbital angular momentum 54 transfer to anisotropic particle 27

28 Setup for Microhologram Rotation microhologram rotating beads collection lens 55 Rotating beads with Microhologram incident laser beam: linearly polarized, Gaussian 56 28

29 UQ Laser tweezers team Studnets: Gregor Knöner Simon Parkin Julien Higuet Justin Ross Cavin Talbot Staff: A/Prof. N. Heckenberg Dr. MArlies Friese Dr. Timo Nieminen Dr. Wolfgang Singer HRD Former Members: Martin Dienstbier Dr. Alexis Bishop 57 Thank you for your attention!!! Simon Parkin Rotation using orbital AM LG 02 doughnut beam 58 29